Cryo-ET of mature and premature axon branches. (A) Low-magnification view of a mature axon branching site. White box depicts area of tomographic data collection shown in B. (B) Slice of the tomographic reconstruction of the branching site. (C) Segmentation of the 430-nm thick tomographic volume from B. Color code: gray, cellular membrane; green, MTs; light blue, actin; pink, mitochondria; yellow, ER; dark blue, ribosomes; orange, vesicles. White arrow shows the direction of bundled MTs following axon growth; white arrowheads depict MTs entering the branch. (D–G) Zoomed-in views of segmented volume: ER wrapping around mitochondrion (white arrowhead; D), ER wrapping around MTs (E), ER forming a flat sheet (white arrowhead; F), ribosomes in the vicinity of mitochondrion (G). (H) Slice from tomographic reconstruction of a premature branch. (I) 353-nm segmented volume of tomogram (H; same color code as in C); white arrowhead depicts filopodium filled with actin. (J) Actin arrangement in the filopodia of a premature branch. (K) Actin arrangement in a mature branch (traced actin in light blue; white arrow shows direction of main axon growth). Scale bars: 500 nm (A); 100 nm (B, C, H, and I); 25 nm (D–G); 50 nm (J and K).